The Ephrin (Eph) receptors are a large family of receptor tyrosine kinases with many functions in physiology and disease. They bind their activating ligands, the ephrins, mainly through a high-affinity binding pocket located in the N-terminal ephrin-binding domain. Each of the five ephrin-A ligands can bind to most of the nine EphA receptors and each of the three ephrin-B ligands can bind to the five EphB receptors. A cysteine-rich region and two fibronectin type III domains connect the ephrin-binding domain to the transmembrane segment. The cytoplasmic portion of the Eph receptors includes a juxtamembrane segment, the kinase domain, a sterile-alpha-motif (SAM) domain and a C-terminal PDZ domain-binding motif. Interaction between Eph receptors and ephrin ligands, which are attached to the cell surface through a GPI-anchor (ephrin-As) or a transmembrane domain (ephrin-Bs), typically occurs at sites of cell-cell contact. Ephrin binding promotes activation of the receptor's kinase domain, triggering “forward” signals. Ephrin ligands engaged with Eph receptors can also affect the cells in which they are expressed by mediating “reverse” signals.
Ephrin type-A receptor 4 (EphA4) signaling can be activated by all ephrin ligands, including the five GPI-linked ephrin-As and the three transmembrane ephrin-Bs. Highly expressed in the nervous system, EphA4 tyrosine kinase activity and downstream signaling leads to inhibition of axon growth and retraction of synaptic structures known as dendritic spines. The repulsive effects of EphA4 in neurons help guide the growth of developing axons towards their synaptic targets and may contribute to inhibition of axon regeneration following injury. In addition, EphA4 interaction with the ephrin-A3 ligand expressed in astrocytes stimulates “reverse” signals through the ephrin that limit the uptake of the extracellular neurotransmitter glutamate, thus modulating synaptic transmission. EphA4 is also highly expressed in adult hippocampal neurons, where it controls synaptic morphology and plasticity. Furthermore, EphA4 appears to contribute to the maintenance of brain neural stem cells in an undifferentiated state. This is in contrast to muscle, where EphA4 may contribute to myoblast differentiation.
Dysregulation of EphA4 activity and/or function has been implicated in the pathophysiology of neurodegenerative disorders, the promotion of neurotoxicity, the inhibition of nerve differentiation and regeneration, and in the progression of cancer. For example, low EphA4 expression and loss-of-function mutations are linked to late onset and prolonged survival in amyotrophic lateral sclerosis (ALS), a fatal diseases that still lack any means for effective therapeutic intervention. Even partial EphA4 gene inactivation has shown beneficial effects in animal models of ALS, making EphA4 inhibition an attractive strategy for counteracting neurodegeneration. In addition, EphA4 was identified as a possible inhibitor of nerve regeneration after spinal cord injury. Experiments in mice suggest a role for EphA4 in the behavioral responses to cocaine administration. Further evidence also supports the involvement of EphA4 in the pathogenesis of spinal cord injury and other neurological diseases such as Alzheimer's disease, multiple sclerosis, stroke and traumatic brain injury. These pathological roles of EphA4 in the diseased nervous system are regarded as being linked to its increased expression and activation by ephrin ligands or Aβ-oligomers in the Alzheimer's brain, leading to abnormal inhibition of axon growth, synaptic function and neuronal survival. Furthermore, EpHA4 signaling prevents the generation of cochlear sensory hair cells suggesting that inhibition of EpHA4 activity could be an effective therapy in the treatment of hearing loss. Finally, increasing evidence also implicates EphA4 in various types of cancer, including glioblastoma, gastric cancer, pancreatic cancer, prostate cancer and breast cancer. For example, EphA4 downregulation studies have suggested a role for EphA4 in leukemia, prostate cancer, pancreatic cancer and gastric cancer cell growth and in liver cancer metastasis. High EphA4 expression has also been correlated with shorter survival in breast and gastric cancer patients, although the opposite correlation was found in lung cancer patients. EphA4 is also highly upregulated in Sezary syndrome, a leukemic variant of cutaneous T-cell lymphomas. Finally, EphA4 can enhance the oncogenic effects of fibroblast growth factor receptor 1 in glioblastoma cells. Hence, inhibiting EphA4-ephrin interaction could be useful for promoting axon regeneration and neural repair, providing neuroprotection and regulating synaptic plasticity in the nervous system as well as inhibiting the progression of cancer.
The two main strategies to block ephrin-induced EphA4 receptor signaling are inhibition of EphA4 kinase activity using kinase inhibitors and inhibition of ephrin binding to the EphA4 ligand binding domain using antagonists. Kinase inhibitors are hampered by low selectivity because they typically target multiple kinases due to the high conservation of the ATP binding pocket. As such, it is very difficult to identify kinase inhibitors selective for EphA4. In contrast, the ephrin-binding pocket in the extracellular EphA4 ligand binding domain has unique features that can be exploited for more selective antagonist targeting. However, the ephrin-binding pocket is very broad (exceeding 900 Å2) and shallow for high affinity binding of small molecules, and small molecule EphA4 antagonists found to date are not very potent and exhibit problematic features that make them unsuitable for therapeutic applications. On the other hand, peptide antagonists have been identified that are highly selective for the ephrin-binding pocket of EphA4. The most potent peptide antagonist identified to date was the linear dodecapeptide KYLPYWPVLSSL (KYL; SEQ ID NO: 1) which was shown to specifically inhibit EphA4 signaling in culture systems and animal models. The KYL peptide significantly dampened ALS pathogenesis in the classic rat SOD1 G93A ALS model. In addition, recent data have shown that KYL peptide can inhibit the toxic effects of Aβ oligomers in in vitro and in vivo mouse models of Alzheimer's disease. The KYL peptide was also shown to promote axon sprouting and recovery of limb function in a rat model of spinal cord injury. Thus, the KYL peptide clearly demonstrated the therapeutic potential of EphA4 antagonistic agents. However, with a KD value of about 800-1000 nM, the linear KYL peptide lacks desired features, and as such, is not ideally suited as a platform for therapeutic development. In addition, both a phage display screen of a cyclic nonapeptide library and an NMR-based screen for smaller EphA4 peptidomimetic antagonists failed to yield peptides more potent than KYL.
Therefore there is still a need to identify EphA4 peptide antagonists that possess the required potency and stability in biological systems to make them suitable therapeutic agents in the treatment of neurodegenerative disorders, neurotoxicity, nerve regeneration and cancer.